Design and statistical optimisation of emulsomal nanoparticles for improved anti-SARS-CoV-2 activity of N-(5-nitrothiazol-2-yl)-carboxamido candidates: in vitro and in silico studies

Abstract In this article, emulsomes (EMLs) were fabricated to encapsulate the N-(5-nitrothiazol-2-yl)-carboxamido derivatives (3a–3g) in an attempt to improve their biological availability and antiviral activity. Next, both cytotoxicity and anti-SARS-CoV-2 activities of the examined compounds loaded EMLs (F3a–g) were assessed in Vero E6 cells via MTT assay to calculate the CC50 and inhibitory concentration 50 (IC50) values. The most potent 3e-loaded EMLs (F3e) elicited a selectivity index of 18 with an IC50 value of 0.73 μg/mL. Moreover, F3e was selected for further elucidation of a possible mode of action where the results showed that it exhibited a combination of virucidal (>90%), viral adsorption (>80%), and viral replication (>60%) inhibition. Besides, molecular docking and MD simulations towards the SARS-CoV-2 Mpro were performed. Finally, a structure–activity relationship (SAR) study focussed on studying the influence of altering the size, type, and flexibility of the α-substituent to the carboxamide in addition to compound contraction on SARS-CoV-2 activity. Highlights Emulsomes (EMLs) were fabricated to encapsulate the N-(5-nitrothiazol-2-yl)-carboxamido derivatives (3a–3g). The most potent 3e-loaded EMLs (F3e) showed an IC50 value of 0.73 μg/mL against SARS-CoV-2. F3e exhibited a combination of virucidal (>90%), viral adsorption (>80%), and viral replication (>60%) inhibition. Molecular docking, molecular dynamics (MD) simulations, and MM-GBSA calculations were performed. Structure–activity relationship (SAR) study was discussed to study the influence of altering the size, type, and flexibility of the α-substituent to the carboxamide on the anti-SARS-CoV-2 activity.


Supplementary Material
Materials and Methods S1: MTT cytotoxicity assay (CC50) Samples were diluted with Dulbecco's Modified Eagle's Medium (DMEM). Stock solutions of the test compounds were prepared in 10 % DMSO in dd H 2 O. The cytotoxic activity of the extracts was tested in Vero-E6 cells by using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method with minor modification 1 . Briefly, the cells were seeded in 96 well-plates (100 µL/well at a density of 3×10 5 cells/mL) and incubated for 24 h at 37 o C in 5% CO2. After 24 h, cells were treated with various concentrations of the tested samples in triplicates. After a further 72 h, the supernatant was discarded and cell monolayers were washed with 1X sterile phosphate buffer saline (PBS) 3 times and MTT solution (20 µL of 5 mg/mL stock solution) was added to each well and incubated at 37 °C for 4 h followed by medium aspiration. In each well, the formed formazan crystals were dissolved with 200 µL of DMSO (0.04 M HCl in absolute isopropanol = 0.073 mL HCl in 50 mL isopropanol). The absorbance of formazan solutions was measured at λmax 540 nm with 620 nm as a reference wavelength using a multi-well plate reader. The plot of % cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (TC50). The % of cytotoxicity compared to the untreated cells was determined with the following equation:

S2: Inhibitory concentration 50 (IC50) determination
The Vero-E6 cells (2.4×104) were kept overnight at 37°C in 5% CO2 inside 96-well tissue culture plates. 1x PBS solution was used to wash the cell monolayers for only one time which were then treated with different serial dilutions of the examined compounds together with a fixed dilution from the virus (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) following TCID50 test and kept at RT for 1 h before starting incubation. Also, the cell monolayers were subjected to DMEM (100 μl) with different concentrations of the test samples and virus and left at 37°C for 72 h in a 5% CO2.
Then, 4% paraformaldehyde (100 μl) was used for cell fixation (2 h) followed by the staining step with 0.1% crystal violet in distilled H2O (50 μl) at RT for 15 min. Absolute CH3OH (100 μl) was added to S3 dissolve the crystal violet dye per well to measure the optical density of the produced color using Anthos Zenyth 200rt plate reader at 570 nm. 2 The IC50 value for each tested compound which is corresponding to its minimum concentration required to reduce the virus infectivity by 50% in comparison to the virus control was calculated.

S3: Mode of action of virus inhibition
The

S3.1. Virucidal
The virucidal assay was carried out 3 in a 6 wells plate where Vero-E6 cells were cultivated (10 5 cells/mL) for 24 h at 37 o C. A volume of 200 µL serum-free DMEM containing virus was added to the concentration of the tested extract. After 1 h incubation, the mixture was diluted using serum-free medium 3 times each 10-fold which still allows the existence of viral particles to grow on Vero-E6 cells but leaves nearly no extract and 100 µL of each dilution was added to the Vero-E6 cell monolayer. After 1 h contact time, DMEM over layer was added to cell monolayer. Plates were left to solidify and then incubated at 37 o C to allow the formation of viral plaques, fixed and stained as above mentioned to calculate percentage reduction in plaques formation in comparison to control wells where cells were infected with the virus that was not pretreated with the tested extract.

S3.2. Viral adsorption
Vero-E6 cells were cultivated in a 6 wells plate (10 5 cells/mL) for 24 h at 37 o C for the viral adsorption assay using the Zhang et al. method 4 . The plant extract was applied at different concentrations in a 200 µL medium without supplements and co-incubated with the cells for 2 h at 4 o C. The unabsorbed extract was removed by washing cells 3 successive times with supplements free-medium then virus diluted was co-incubated with the pretreated cells for 1 h followed by adding 3 mL DMEM supplemented with S4 2% agarose. Plates were left to solidify and then incubated at 37 o C to allow the formation of viral plaques, fixed and stained as above mentioned to calculate percentage reduction in plaques formation in comparison to control wells where untreated Vero-E6 cells were directly infected with the virus.

S3.3. Viral replication
The viral replication assay was carried out according to Kuo et al. 5  Cell monolayers were fixed in 10% formalin solution for 2 h, and stained with crystal violet. Control wells were included where Vero-E6 cells were incubated with the virus and didn't treat with the extract. Finally, plaques were counted and percentage reduction in plaques formation in comparison to control wells was recorded as above mentioned.

S4: Molecular dynamics simulations
The MD simulations were carried out using Desmond simulation package of Schrödinger LLC. 6 The NPT ensemble with the temperature 300 K and a pressure 1 bar was applied in all runs. The simulation length was 200 ns with a relaxation time 1 ps for the ligands. The OPLS3 force field parameters were used in all simulations. 7 The cutoff radius in Coulomb interactions was 9.0 Å. The orthorhombic periodic box boundaries were set 10 Å away from the protein atoms. The water molecules were explicitly described using the transferable intermolecular potential with three points (TIP3P) model. 8,9 Salt concentration set to 0.15 M NaCl and was built using the System Builder utility of Desmond. 10 Figure S1: The RMSF of the proteins C within the binding pocket of SARS-CoV-2 Mpro (PDB ID: 6Y2G) during the simaultion time for compounds 3a, 3b, 3c, 3d, 3e, 3f, 3g, and Co.